Tumor antigen primed dendritic cells (DCs) are under widespread clinical evaluation for immunotherapy of multiple forms of cancer. Emerging evidence suggests that DCs are fundamental regulators of the immune response leading to potent cell mediated immunity or alternatively to negative regulation and tolerance induction. Conventional cytokine combinations and DC culture methodologies widely used to generate cancer vaccines for these human clinical trials produce a mixture of immunoactivating and immunoinhibitory DC subsets as defined by expression and mechanistic association of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). Aastrom Biosciences, Inc. has developed a novel clinical scale bioreactor system for production, antigen loading, maturation and harvest of monocyte-derived dendritic cells using closed system automation and continuous single-pass medium perfusion. The primary goal of this Phase I proposal is to enhance the immunostimulatory potency of dendritic cell based cancer vaccines by removal of immunosuppressive DC subsets while selectively enriching for activating IDO negative DC subsets at the time of harvest from the AastromReplicell TM Cell Production System. A procedure for fractionation of DC subpopulations to high purity based on differential plastic adherence of IDO-negative DCs to the bioreactor cell bed at the time of harvest will be developed, evaluated and automated. New maturation cytokine combinations which specifically promote differentiation of highly immunoactivating DCs while minimizing production of potentially tolerogenic DCs will be identified and evaluated under perfusion culture conditions. Well-defined and clinically applicable cytomegaloviral (CMV)and tumor-associated carcinoembryonic antigen (CEA) peptide epitopes will be used to characterize theCD4+ and CD8+ T-cell responses to these DC subpopulations in vitro as a prerequisite to evaluation of the vaccine for safety, immunological and clinical efficacy for immunotherapy of human patients with CEA positive malignancies at Duke University Medical Center and other clinical sites during the Phase II SBIR proposal. A closed automated bioreactor system will fulfill a large unmet clinical demand for consistent, reliable and reproducible DC vaccine production under stringent regulatory conditions with improved immunologic and therapeutic potency for immunotherapy of infectious diseases and cancer.